Modulators of fatty acid amide hydrolase

ABSTRACT

4-(2,2-Difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylic acid (4-chloro-pyridin-3-yl)-amide is described, which is useful as a FAAH modulator. 4-(2,2-Difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylic acid (4-chloro-pyridin-3-yl)-amide may be used in pharmaceutical compositions and methods for the treatment of disease states, disorders, and conditions mediated by fatty acid amide hydrolase (FAAH) activity, such as anxiety, pain, inflammation, sleep disorders, eating disorders, energy metabolism disorders, and movement disorders (e.g., multiple sclerosis). A method of synthesizing 4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylic acid (4-chloro-pyridin-3-yl)-amide is also disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application is a national phase of International Application No.PCT/US2011/034755 filed May 2, 2011, and claims benefit of priority ofU.S. Provisional Application No. 61/330,522 filed on May 3, 2010.

FIELD OF THE INVENTION

The present invention relates to the compound4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide, pharmaceutical compositionscontaining the compound, methods of synthesizing the compound, andmethods of using the compound for the treatment of disease states,disorders, and conditions mediated by fatty acid amide hydrolase (FAAH)activity are provided.

BACKGROUND OF THE INVENTION

Medicinal benefits have been attributed to the cannabis plant forcenturies. The primary bioactive constituent of cannabis isΔ⁹-tetrahydro-cannabinol (THC). The discovery of THC eventually led tothe identification of two endogenous cannabinoid receptors responsiblefor its pharmacological actions, namely CB₁ and CB₂ (Goya et al., Exp.Opin. Ther. Patents, 2000, 10, 1529). These discoveries not onlyestablished the site of action of THC, but also inspired inquiries intothe endogenous agonists of these receptors, or “endocannabinoids”. Thefirst endocannabinoid identified was the fatty acid amide arachidonylethanolamide or anandamide (AEA). AEA itself elicits many of thepharmacological effects of exogenous cannabinoids (Piomelli et al., Nat.Rev. Neurosci., 2003, 4(11), 873).

The catabolism of AEA is primarily attributable to the integral membranebound protein fatty acid amide hydrolase (FAAH), which hydrolyzes AEA toarachidonic acid and ethanolamine. FAAH was characterized in 1996 byCravatt and co-workers (Cravatt et al., Nature, 1996, 384, 83). It wassubsequently determined that FAAH is additionally responsible for thecatabolism of a large number of important lipid signaling fatty acidamides including: another major endocannabinoid, 2-arachidonoylglycerol(2-AG) (Devane et al., Science, 1992, 258, 1946-1949); thesleep-inducing substance, oleamide (Cravatt et al., Science, 1995, 268,1506); the appetite-suppressing agent, N-oleoylethanolamide (OEA)(Rodriguez de Fonesca, Nature, 2001, 414, 209); and theanti-inflammatory agent, palmitoylethanolamide (PEA) (Lambert et al.,Curr. Med. Chem., 2002, 9(6), 663).

Small-molecule inhibitors of FAAH should elevate the concentrations ofthese endogenous signaling lipids and thereby produce their associatedbeneficial pharmacological effects. There have been some reports of theeffects of various FAAH inhibitors in pre-clinical models.

In particular, two carbamate-based inhibitors of FAAH were reported tohave analgesic properties in animal models. In rats, BMS-1 (see WO02/087569), which has the structure shown below, was reported to have ananalgesic effect in the spinal nerve ligation (Chung) model ofneuropathic pain, and the Hargreaves test of acute thermal nociception.URB-597 was reported to have efficacy in the zero plus maze model ofanxiety in rats, as well as analgesic efficacy in the rat hot plate andformalin tests (Kathuria et al., Nat. Med., 2003, 9(1), 76). The urea,4-(3-Phenyl-[1,2,4]thiadiazol-5-yl)-piperazine-1-carboxylic acidphenylamide, was found to be efficacious in both the spinal nerveligation (Chung) model of neuropathic pain and the mild thermal injurymodel of acute burn injury pain (Karbarz et al., Anesth Analg., 2009,108(1), 316-329). Other potent urea inhibitors of the FAAH enzyme havebeen reported (WO 06/074025). The sulfonylfluoride AM374 was also shownto significantly reduce spasticity in chronic relapsing experimentalautoimmune encephalomyelitis (CREAE) mice, an animal model of multiplesclerosis (Baker et al., FASEB J., 2001, 15(2), 300).

In addition, the oxazolopyridine ketone OL-135 is reported to be apotent inhibitor of FAAH with analgesic activity in both the hot plateand tail immersion tests of thermal nociception in rats (WO 04/033652).

Results of research on the effects of certain exogenous cannabinoids haselucidated that a FAAH inhibitor may be useful for treating variousconditions, diseases, disorders, or symptoms. These include pain,nausea/emesis, anorexia, spasticity, movement disorders, epilepsy andglaucoma. To date, approved therapeutic uses for cannabinoids includethe relief of chemotherapy-induced nausea and emesis among patients withcancer and appetite enhancement in patients with HIV/AIDs who experienceanorexia as a result of wasting syndrome. Two products are commerciallyavailable in some countries for these indications, namely, dronabinol(Marinol®) and nabilone.

Apart from the approved indications, a therapeutic field that hasreceived much attention for cannabinoid use is analgesia, i.e., thetreatment of pain. Five small randomized controlled trials showed thatTHC is superior to placebo, producing dose-related analgesia (Robson etal., Br. J. Psychiatry, 2001, 178, 107-115). Atlantic Pharmaceuticals isreported to be developing a synthetic cannabinoid, CT-3, a 1,1-dimethylheptyl derivative of the carboxylic metabolite of tetrahydrocannabinol,as an orally active analgesic and anti-inflammatory agent. A pilot phaseII trial in chronic neuropathic pain with CT-3 was reportedly initiatedin Germany in May 2002.

A number of individuals with locomotor activity-related diseases, suchas multiple sclerosis have claimed a benefit from cannabis for bothdisease-related pain and spasticity, with support from small controlledtrials (Croxford et el., J. Neuroimmunol, 2008, 193, 120-9; Svendsen,Br. Med. J., 2004, 329, 253). Likewise, various victims of spinal cordinjuries, such as paraplegia, have reported that their painful spasmsare alleviated after smoking marijuana. A report showing thatcannabinoids appear to control spasticity and tremor in the CREAE modelof multiple sclerosis demonstrated that these effects are mediated byCB₁ and CB₂ receptors (Baker, Nature, 2000, 404, 84-87). Phase 3clinical trials have been undertaken in multiple sclerosis and spinalcord injury patients with a narrow ratio mixture oftetrahydrocannabinol/cannabidiol (THC/CBD). It has been reported thatFAAH knockout mice consistently recover to a better clinical score thanwild type controls, and this improvement is not a result ofanti-inflammatory activity, but rather may reflect some neuroprotectionor remyelination promoting effect of lack of the enzyme (Webb et al,Neurosci Lett., 2008, vol. 439, 106-110).

Reports of small-scale controlled trials to investigate other potentialcommercial uses of cannabinoids have been made. Trials in volunteershave been reported to have confirmed that oral, injected, and smokedcannabinoids produced dose-related reductions in intraocular pressure(IOP) and therefore may relieve glaucoma symptoms. Ophthalmologists haveprescribed cannabis for patients with glaucoma in whom other drugs havefailed to adequately control intraocular pressure (Robson et al., 2001,supra).

Inhibition of FAAH using a small-molecule inhibitor may be advantageouscompared to treatment with a direct-acting CB₁ agonist. Administrationof exogenous CB₁ agonists may produce a range of responses, includingreduced nociception, catalepsy, hypothermia, and increased feedingbehavior. These four in particular are termed the “cannabinoid tetrad.”Experiments with FAAH^(−/−) mice show reduced responses in tests ofnociception, but did not show catalepsy, hypothermia, or increasedfeeding behavior (Cravatt et al., Proc. Natl. Acad. Sci. USA, 2001,98(16), 9371). Fasting caused levels of AEA to increase in rat limbicforebrain, but not in other brain areas, providing evidence thatstimulation of AEA biosynthesis may be anatomically regionalized totargeted CNS pathways (Kirkham et al., Br. J. Pharmacol., 2002, 136,550). The finding that AEA increases are localized within the brain,rather than systemic, suggests that FAAH inhibition with a smallmolecule could enhance the actions of AEA and other fatty acid amides intissue regions where synthesis and release of these signaling moleculesis occurring in a given pathophysiological condition (Piomelli et al.,2003, supra).

In addition to the effects of a FAAH inhibitor on AEA and otherendocannabinoids, inhibitors of FAAH's catabolism of other lipidmediators may be used in treating certain other therapeutic indications.For example, PEA has demonstrated biological effects in animal models ofinflammation (Holt et al., Br. J. Pharmacol., 2005, 146, 467-476),immunosuppression, analgesia, and neuroprotection (Ueda et al., J. Biol.Chem., 2001, 276(38), 35552). Oleamide, another substrate of FAAH,induces sleep (Boger et al., Proc. Natl. Acad. Sci. USA, 2000, 97(10),5044; Mendelson et al., Neuropsychopharmacology, 2001, 25, S36).Inhibition of FAAH has also been implicated in cognition (Varvel et al.,J. Pharmacol. Exp. Ther., 2006, 317(1), 251-257) and depression (Gobbiet al., Proc. Natl. Acad. Sci. USA, 2005, 102(51), 18620-18625).

Two additional indications for FAAH are supported by recent dataindicating that FAAH substrate activated receptors are important inenergy metabolism, and in bone homeostasis (Overton et al., Br. J.Pharmacol., 2008, 153 Suppl 1, S76-81; and Plutzky et al., Diab. Vasc.Dis. Res., 2007, 4 Suppl 3, S12-4). It has been shown that one of thepreviously mentioned lipid signaling fatty acid amides catabolized byFAAH, OEA, is one of the most active agonists of the recentlyde-orphanised GPCR 119 (GPR119) (also termed glucose-dependentinsulinotropic receptor). This receptor is expressed predominantly inthe pancreas in humans and activation improves glucose homeostasis viaglucose-dependent insulin release in pancreatic beta-cells. GPR119agonists can suppress glucose excursions when administered during oralglucose tolerance tests, and OEA has also been shown independently toregulate food intake and body weight gain when administered to rodents,indicating a probable benefit in energy metabolism disorders, such asinsulin resistance and diabetes. The FAAH substrate PEA is an agonist atthe PPARα receptor. Evidence from surrogate markers in human studieswith the PPARα agonist fenofibrate is supportive of the concept thatPPARα agonism offers the potential for inducing a coordinated PPARαresponse that may improve dyslipidaemia, repress inflammation and limitatherosclerosis in patients with the metabolic syndrome or type 2diabetes. Anandamide is an agonist at the PPARγ receptor. Anandamidetreatment induces 3T3-L1 differentiation into adipocytes, as well astriglyceride droplet accumulation and expression of adiponectin(Bouaboula et al., E. J. Pharmacol., 2005, 517, 174-181). Low dosecannabinoid therapy has been shown to reduce atherosclerosis in mice,further suggesting a therapeutic benefit of FAAH inhibition indyslipidemia, liver steatosis, steatohepatitis, obesity, and metabolicsyndrome (Steffens et al., Nature, 2005, 434, 782-6).

Osteoporosis is one of the most common degenerative diseases. It ischaracterized by reduced bone mineral density (BMD) with an increasedrisk for bone fractures. CB₂-deficient mice have a markedly acceleratedage-related trabecular bone loss and cortical expansion. A CB₂-selectiveagonist enhances endocortical osteoblast number and activity andrestrains trabecular osteoclastogenesis and attenuatesovariectomy-induced bone loss (Ofek et al., Proc. Natl. Acad. Sci.U.S.A., 2006, 103, 696-701). There is a substantial genetic contributionto BMD, although the genetic factors involved in the pathogenesis ofhuman osteoporosis are largely unknown. The applicability to human BMDis suggested by genetic studies in which a significant association ofsingle polymorphisms and haplotypes was found encompassing the CNR2 geneon human chromosome 1p36, demonstrating a role for the peripherallyexpressed CB₂ receptor in the etiology of osteoporosis (Karsak et al.,Hum. Mol. Genet, 2005, 14, 3389-96).

Thus, small-molecule FAAH inhibitors should be useful in treating painof various etiologies, anxiety, multiple sclerosis and other movementdisorders, nausea/emesis, eating disorders, epilepsy, glaucoma,inflammation, immunosuppression, neuroprotection, depression, cognitionenhancement, and sleep disorders, and potentially with fewer sideeffects than treatment with an exogenous cannabinoid.

A number of heteroaryl-substituted ureas have been reported in variouspublications. Certain piperazinyl and piperidinyl compounds as FAAHmodulators are described in Intl. Patent Appl. No. WO 2006/074025, Intl.Patent Appl. Ser. No. PCT/US2009/065757, Intl. Patent Appl. Ser. No.PCT/US2009/065752, U.S. Appl. Publ. No. US 2009/0062294, and U.S.provisional Appl. Ser. No. 61/263,477. Certain piperazine-1-carboxamideand piperidine-1-carboxamide derivatives are described in Intl. PatentAppl. No. WO 2008/023720. Certain aryloxobutylpiperidines,aryloxobutylpyrrolidines, and aryloxobutylpiperazines are described inIntl. Patent Appl. No. WO 2001/005763. Certain piperidine derivativesare reported in Intl. Patent Appl. No. WO 99/50247. Certain piperazinederivatives are described in Intl. Patent Appl. No. WO 99/42107. CertainN-aralkylpiperazines are described in Intl. Patent Appl. No. WO98/37077. Certain aryl-substituted heterocyclic urea derivatives aredescribed in U.S. provisional Appl. No. 61/184,606. However, thereremains a desire for potent FAAH modulators with suitable pharmaceuticalproperties.

The features and advantages of the present invention are apparent to oneof ordinary skill in the art. Based upon this disclosure, including thesummary, detailed description, background, examples, and claims, one ofordinary skill in the art will be able to make modifications andadaptations to various conditions and usages. Publications describedherein are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

4-(2,2-Difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide, and pharmaceutically acceptablesalts thereof, are herein described, which have been found to haveFAAH-modulating activity. The invention is directed to the general andpreferred embodiments defined, respectively, and by the independent anddependent claims appended hereto, which are incorporated by referenceherein.

The present invention provides experimental evidence demonstrating thatthe chemical entities of the present invention exhibit higher 1050values in CYP2D6 inhibition when compared to a comparator compound.Additionally,4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide exhibited improved behavior andphysiological function side effects as a result of compoundadministration when compared to comparator compound in a primaryobservation (Irwin) test in rats.

The 4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide IC₅₀ activity of CYP2D6 inhibition isimproved in comparison to a previously described piperazinyl ureacompound,4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid pyridin-3-ylamide (see PCT. Pub. Appl. No. WO 2006/074025, example150), which is herein described as a comparator compound.4-(2,2-Difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide exhibited an IC₅₀ value 7.5 to 5.5times higher versus the comparator compound with bufuralol ordextromethorphan being used as substrates, respectively.

Furthermore, the chemical entities of the present invention displayunpredicted characteristics in a primary observation (Irwin) test inrats when compared to the comparator compound. In particular, comparatorcompound4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid pyridin-3-ylamide increased reactivity to touch in all rats testedat the 10 mg/kg dose while4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide induced reactivity in only one offour rats tested at the same dosage. The comparator compound inducedsedation at the 15 to 120 minute interval and abnormal gait (rolling) at15 minutes post treatment in all rats tested at 60 mg/kg while suchobservations were not apparent in4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide treated rats. Also apparent at the 60mg/kg dosage, the comparator compound decreased muscle tone for all ratsin the 60 to 120 minute time interval while4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide increased abdominal muscle tome inonly one of four rats tested. Finally, at 60 mg/kg dosing the comparatorcompound induced hypothermia in rat subjects at 15 to 60 minute and 180minute intervals while this observation was absent in rats tested withthe compound of the present invention.

In one general aspect, the invention is directed to the compound of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide. In a particular embodiment, thecompound is a hydrochloride salt of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide.

The invention also relates to pharmaceutically acceptable salts of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide, pharmaceutically acceptable prodrugsof 4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide, and pharmaceutically acceptablemetabolites of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide.

In a further general aspect, the invention relates to pharmaceuticalcompositions each comprising: (a) a therapeutically effective amount ofat least one of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide, pharmaceutically acceptable salts of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide, pharmaceutically acceptable prodrugsof 4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide, and pharmaceutically acceptablemetabolites of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide; and (b) a pharmaceuticallyacceptable excipient.

In another aspect, embodiments of the invention are useful as FAAHmodulators. Thus, the invention is directed to a method for modulatingFAAH activity, comprising exposing FAAH to a therapeutically effectiveamount of at least one of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide, pharmaceutically acceptable salts of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethylypiperazine-1-carboxylic acid(4-chloro-pyridin-3-yl)-amide, pharmaceutically acceptable prodrugs of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide, and pharmaceutically activemetabolites of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide.

In another general aspect, the invention is directed to a method oftreating a subject suffering from or diagnosed with a disease, disorder,or medical condition mediated by fatty acid amide hydrolase (FAAH)activity, comprising administering to the subject in need of suchtreatment an effective amount of at least one agent selected from4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide and its pharmaceutically acceptablesalts, pharmaceutically active prodrugs, and pharmaceutically activemetabolites. In preferred embodiments of the inventive method, thedisease, disorder, or medical condition is selected from: anxiety,depression, pain, sleep disorders, eating disorders, inflammation,multiple sclerosis and other movement disorders, HIV wasting syndrome,closed head injury, stroke, learning and memory disorders, Alzheimer'sdisease, epilepsy, Tourette's syndrome, Niemann-Pick disease,Parkinson's disease, Huntington's chorea, optic neuritis, autoimmuneuveitis, symptoms of drug or alcohol withdrawal, nausea, emesis, sexualdysfunction, anxiety, post-traumatic stress disorder, cerebralvasospasm, glaucoma, irritable bowel syndrome, inflammatory boweldisease, immunosuppression, itch, gastroesophageal reflux disease,paralytic ileus, secretory diarrhea, gastric ulcer, rheumatoidarthritis, unwanted pregnancy, hypertension, cancer, hepatitis, allergicairway disease, auto-immune diabetes, intractable pruritis,neuroinflammation, diabetes, metabolic syndrome, osteoporosis,dyslipidemia, liver steatosis, and steatohepatitis.

In another general aspect, the invention is directed towards a method ofsynthesizing4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide using2,2-difluoro-benzo[1,3]dioxole-5-carbaldehyde and piperazine in a singlestep hydrogenation reaction.

Additional embodiments, features, and advantages of the invention willbe apparent from the following detailed description and through practiceof the invention.

DETAILED DESCRIPTION OF THE INVENTION AND ITS PREFERRED EMBODIMENTS

The invention may be more fully appreciated by reference to thefollowing description, including the following glossary of terms and theconcluding examples. For the sake of brevity, the disclosures of thepublications cited in this specification are herein incorporated byreference.

As used herein, the terms “including”, “containing” and “comprising” areused herein in their open, non-limiting sense.

A structural formula given herein is also intended to representunlabeled forms as well as isotopically labeled forms of the compounds.Isotopically labeled compounds have structures depicted by the formulasgiven herein except that one or more atoms are replaced by an atomhaving a selected atomic mass or mass number. Examples of isotopes thatcan be incorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, or fluorine, such as ²H, ³H, ¹¹C,¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, and ¹⁸F, respectively. Such isotopicallylabeled compounds are useful in metabolic studies (preferably with ¹⁴C),reaction kinetic studies (with, for example ²H or ³H), detection orimaging techniques [such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT), including drug orsubstrate tissue distribution assays, or in radioactive treatment ofpatients. In particular, an ¹⁸F- or ¹¹C-labeled compound may bepreferred for PET or SPECT studies. Further, substitution with heavierisotopes such as deuterium (i.e., ²H) may afford certain therapeuticadvantages resulting from greater metabolic stability, for example,increased in vivo half-life or reduced dosage requirements. Isotopicallylabeled compounds of this invention and prodrugs thereof can generallybe prepared by carrying out the procedures disclosed in the schemes orin the examples and preparations described below by substituting areadily available isotopically labeled reagent for a non-isotopicallylabeled reagent.

In one general embodiment, the invention relates to4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide and pharmaceutically acceptablesalts, pharmaceutically acceptable prodrugs, and pharmaceutically activemetabolites of such a compound. In another general embodiment, theinvention relates to pharmaceutical compositions each comprising atherapeutically effective amount of a FAAH-modulating agent selectedfrom4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide and pharmaceutically acceptablesalts, pharmaceutically acceptable prodrugs, and pharmaceutically activemetabolites of such a compound.

The invention also relates to pharmaceutically acceptable salts of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide. A “pharmaceutically acceptable salt”is intended to mean a salt of a free acid or base of a compound of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide that is non-toxic, biologicallytolerable, or otherwise biologically suitable for administration to thesubject. See, generally, G. S. Paulekuhn, et al., “Trends in ActivePharmaceutical Ingredient Salt Selection based on Analysis of the OrangeBook Database”, J. Med. Chem., 2007, 50:6665-72, S. M. Berge, et al.,“Pharmaceutical Salts”, J Pharm Sci., 1977, 66:1-19, and Handbook ofPharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth,Eds., Wiley-VCH and VHCA, Zurich, 2002. Preferred pharmaceuticallyacceptable salts are those that are pharmacologically effective andsuitable for contact with the tissues of patients without unduetoxicity, irritation, or allergic response. Examples of pharmaceuticallyacceptable salts include sulfates, pyrosulfates, bisulfates, sulfites,bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates,metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates,propionates, decanoates, caprylates, acrylates, formates, isobutyrates,caproates, heptanoates, propiolates, oxalates, malonates, succinates,suberates, sebacates, fumarates, maleates, butyne-1,4-dioates,hexyne-1,6-dioates, benzoates, chlorobenzoates, methyl benzoates,dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates,sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates,tartrates, methane-sulfonates, propanesulfonates,naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.

In certain embodiments, the compound,4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide, is a hydrochloride salt.

A compound of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide may possess a sufficiently basicgroup and accordingly may react with a number of inorganic and organicacids, to form a pharmaceutically acceptable salt.

The compound of the invention contains at least one basic nitrogen,therefore, a desired pharmaceutically acceptable salt may be prepared byany suitable method available in the art, for example, by treatment ofthe free base with an inorganic acid, such as hydrochloric acid,hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, boric acid,phosphoric acid, and the like; or with an organic acid, such as aceticacid, phenylacetic acid, propionic acid, stearic acid, lactic acid,ascorbic acid, maleic acid, hydroxymaleic acid, isethionic acid,succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid,oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid,lauric acid, a pyranosidyl acid, such as glucuronic acid or galacturonicacid, an alpha-hydroxy acid, such as mandelic acid, citric acid, ortartaric acid; an amino acid, such as aspartic acid or glutamic acid; anaromatic acid, such as benzoic acid, 2-acetoxybenzoic acid, naphthoicacid, or cinnamic acid; a sulfonic acid, such as laurylsulfonic acid,p-toluenesulfonic acid, methanesulfonic acid, or ethanesulfonic acid; orany other acid and mixture thereof that are regarded as equivalents oracceptable substituents in light of the ordinary level of skill in thisart.

The invention also relates to pharmaceutically acceptable prodrugs of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide. The term “prodrug” means a precursorof a designated compound that, following administration to a subject,yields the compound in vivo via a chemical or physiological process suchas solvolysis or enzymatic cleavage, or under physiological conditions(e.g., a prodrug on being brought to physiological pH is converted tothe compound of Example 1). A “pharmaceutically acceptable prodrug” is aprodrug that is non-toxic, biologically tolerable, and otherwisebiologically suitable for administration to the subject. Illustrativeprocedures for the selection and preparation of suitable prodrugderivatives are described, for example, in “Design of Prodrugs”, ed. H.Bundgaard, Elsevier, 1985.

Examples of prodrugs include compounds having an amino acid residue, ora polypeptide chain of two or more (e.g., two, three or four) amino acidresidues, covalently joined through an amide or ester bond to a freeamino group of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide. Examples of amino acid residuesinclude the twenty naturally occurring amino acids, commonly designatedby three letter symbols, as well as 4-hydroxyproline, hydroxylysine,demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine,gamma-aminobutyric acid, citrulline homocysteine, homoserine, ornithineand methionine sulfone. Additional types of prodrugs may be produced,for instance, by derivatizing free amines as amides, sulfonamides orphosphonamides.

The present invention also relates to pharmaceutically activemetabolites of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide. A “pharmaceutically activemetabolite” means a pharmacologically active product of metabolism inthe body of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide or a salt thereof. Prodrugs andactive metabolites of a compound may be determined using routinetechniques known or available in the art. See, e.g., Bertolini et al.,J. Med. Chem. 1997, 40, 2011-2016; Shan et al., J. Pharm. Sci. 1997, 86(7), 765-767; Bagshawe, Drug Dev. Res. 1995, 34, 220-230; Bodor, Adv.Drug Res. 1984, 13, 224-331; Bundgaard, Design of Prodrugs (ElsevierPress, 1985); and Larsen, Design and Application of Prodrugs, DrugDesign and Development (Krogsgaard-Larsen et al., eds., Harwood AcademicPublishers, 1991).

A compound of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide, and its pharmaceutically acceptablesalts, pharmaceutically acceptable prodrugs, and pharmaceutically activemetabolites (collectively, “active agents”) of the present invention areuseful as FAAH inhibitors in the methods of the invention. The term“inhibitors” refers to compounds that decrease, prevent, inactivate,desensitize or down-regulate FAAH expression or activity. The activeagents may be used in the inventive methods for the treatment of medicalconditions, diseases, or disorders mediated through inhibition ormodulation of FAAH, such as those described herein. Active agentsaccording to the invention may therefore be used as an analgesic,anti-depressant, cognition enhancer, neuroprotectant, sedative, appetitestimulant/suppressor, or contraceptive.

Exemplary medical conditions, diseases, and disorders mediated by FAAHactivity include anxiety, depression, pain, sleep disorders, eatingdisorders, inflammation, multiple sclerosis and other movementdisorders, HIV wasting syndrome, closed head injury, stroke, learningand memory disorders, Alzheimer's disease, epilepsy, Tourette'ssyndrome, epilepsy, Niemann-Pick disease, Parkinson's disease,Huntington's chorea, optic neuritis, autoimmune uveitis, symptoms ofdrug or alcohol withdrawal, nausea, emesis, sexual dysfunction,post-traumatic stress disorder, cerebral vasospasm, diabetes, metabolicsyndrome, osteoarthritis and osteoporosis.

Thus, the active agents may be used to treat subjects diagnosed with orsuffering from such a disease, disorder, or condition. The term “treat”or “treating” as used herein is intended to refer to administration ofan agent or composition of the invention to a subject for the purpose ofeffecting a therapeutic benefit through inhibition of FAAH activity.Treating includes reversing, ameliorating, alleviating, inhibiting theprogress of, lessening the severity of, or reducing the incidence of adisease, disorder, or condition, or one or more symptoms of suchdisease, disorder or condition mediated through modulation of FAAHactivity. The term “subject” refers to a mammalian patient in need ofsuch treatment, such as a human. “Modulators” include both inhibitorsand activators, where “inhibitors” refer to compounds that decrease,prevent, inactivate, desensitize or down-regulate FAAH expression oractivity, and “activators” are compounds that increase, activate,facilitate, sensitize, or up-regulate FAAH expression or activity.

Accordingly, the invention relates to methods of using the active agentsdescribed herein to treat subjects diagnosed with or suffering from adisease, disorder, or condition mediated through FAAH activity, such as:anxiety, pain, sleep disorders, eating disorders, inflammation, movementdisorders (e.g., multiple sclerosis), glucose and lipid metabolism (e.g.diabetes) and bone homeostasis (e.g. osteoporosis).

Symptoms or disease states are intended to be included within the scopeof “medical conditions, disorders, or diseases.” For example, pain maybe associated with various diseases, disorders, or conditions, and mayinclude various etiologies. Illustrative types of pain treatable with aFAAH-modulating agent, in one example herein a FAAH-inhibiting agent,according to the invention include cancer pain, postoperative pain, GItract pain, spinal cord injury pain, visceral hyperalgesia, thalamicpain, headache (including stress headache and migraine), low back pain,neck pain, musculoskeletal pain, peripheral neuropathic pain, centralneuropathic pain, neurogenerative disorder related pain, and menstrualpain. HIV wasting syndrome includes associated symptoms such as appetiteloss and nausea. Parkinson's disease includes, for example,levodopa-induced dyskinesia. Treatment of multiple sclerosis may includetreatment of symptoms such as spasticity, neurogenic pain, central pain,or bladder dysfunction. Symptoms of drug withdrawal may be caused by,for example, addiction to opiates or nicotine. Nausea or emesis may bedue to chemotherapy, postoperative, or opioid related causes. Treatmentof sexual dysfunction may include improving libido or delayingejaculation. Treatment of cancer may include treatment of glioma. Sleepdisorders include, for example, sleep apnea, insomnia, and disorderscalling for treatment with an agent having a sedative or narcotic-typeeffect. Eating disorders include, for example, anorexia or appetite lossassociated with a disease such as cancer or HIV infection/AIDS.

In treatment methods according to the invention, an effective amount ofat least one active agent according to the invention is administered toa subject suffering from or diagnosed as having such a disease,disorder, or condition. A “therapeutically effective amount” or“effective amount” means an amount or dose of a FAAH-modulating agentsufficient to generally bring about a desired therapeutic benefit inpatients in need of treatment for a disease, disorder, or conditionmediated by FAAH activity. Effective amounts or doses of the activeagents of the present invention may be ascertained by routine methodssuch as modeling, dose escalation studies or clinical trials, and bytaking into consideration routine factors, e.g., the mode or route ofadministration or drug delivery, the pharmacokinetics of the agent, theseverity and course of the disease, disorder, or condition, thesubject's previous or ongoing therapy, the subject's health status andresponse to drugs, and the judgment of the treating physician. Anexemplary dose is in the range of from about 0.0001 to about 200 mg ofactive agent per kg of subject's body weight per day, preferably about0.001 to 100 mg/kg/day, or about 0.01 to 35 mg/kg/day, or about 0.1 to10 mg/kg daily in single or divided dosage units (e.g., BID, TID, QID).For a 70-kg human, an illustrative range for a suitable dosage amount isfrom about 0.05 to about 7 g/day, or about 0.2 to about 5 g/day. Onceimprovement of the patient's disease, disorder, or condition hasoccurred, the dose may be adjusted for maintenance treatment. Forexample, the dosage or the frequency of administration, or both, may bereduced as a function of the symptoms, to a level at which the desiredtherapeutic effect is maintained. Of course, if symptoms have beenalleviated to an appropriate level, treatment may cease. Patients may,however, require intermittent treatment on a long-term basis upon anyrecurrence of symptoms.

In addition, the active agents of the invention may be used incombination with additional active ingredients in the treatment of theabove conditions. The additional active ingredients may beco-administered separately with an active agent of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide, or included with such an agent in apharmaceutical composition according to the invention. In an exemplaryembodiment, additional active ingredients are those that are known ordiscovered to be effective in the treatment of conditions, disorders, ordiseases mediated by FAAH activity, such as another FAAH modulator or acompound active against another target associated with the particularcondition, disorder, or disease. The combination may serve to increaseefficacy (e.g., by including in the combination a compound potentiatingthe potency or effectiveness of an active agent according to theinvention), decrease one or more side effects, or decrease the requireddose of the active agent according to the invention. In one illustrativeembodiment, a composition according to the invention may contain one ormore additional active ingredients selected from opioids, non-steroidalanti-inflammatory drugs (NSAID) (e.g., ibuprofen, cyclooxygenase-2(COX-2) inhibitors, and naproxen), gabapentin, pregabalin, tramadol,acetaminophen, and aspirin.

The active agents of the invention are used, alone or in combinationwith one or more additional active ingredients, to formulatepharmaceutical compositions of the invention. A pharmaceuticalcomposition of the invention comprises: (a) an effective amount of atleast one active agent in accordance with the invention; and (b) apharmaceutically acceptable excipient.

A “pharmaceutically acceptable excipient” refers to a substance that isnon-toxic, biologically tolerable, and otherwise biologically suitablefor administration to a subject, such as an inert substance, added to apharmacological composition or otherwise used as a vehicle, carrier, ordiluent to facilitate administration of a agent and that is compatibletherewith. Certain pharmaceutically acceptable excipients are reviewedin “Handbook of Pharmaceutical Excipients”, 6^(th) ed., PharmaceuticalPress, 2009. Examples of excipients include calcium carbonate, calciumphosphate, various sugars and types of starch, cellulose derivatives,gelatin, vegetable oils, and polyethylene glycols.

Delivery forms of the pharmaceutical compositions containing one or moredosage units of the active agents may be prepared using suitablepharmaceutical excipients and compounding techniques known or thatbecome available to those skilled in the art. The compositions may beadministered in the inventive methods by a suitable route of delivery,e.g., oral, parenteral, rectal, topical, or ocular routes, or byinhalation.

The preparation may be in the form of tablets, capsules, sachets,dragees, powders, granules, lozenges, powders for reconstitution, liquidpreparations, or suppositories. Preferably, the compositions areformulated for intravenous infusion, topical administration, or oraladministration.

For oral administration, the active agents of the invention can beprovided in the form of tablets or capsules, or as a solution, emulsion,or suspension. To prepare the oral compositions, the active agents maybe formulated to yield a dosage of, e.g., from about 5 mg to 5 g daily,or from about 50 mg to 5 g daily, in single or divided doses. Forexample, a total daily dosage of about 5 mg to 5 g daily may beaccomplished by dosing once, twice, three, or four times per day.

Oral tablets may include the active ingredient(s) mixed with compatiblepharmaceutically acceptable excipients such as diluents, disintegratingagents, binding agents, lubricating agents, sweetening agents, flavoringagents, coloring agents and preservative agents. Suitable inert fillersinclude sodium and calcium carbonate, sodium and calcium phosphate,lactose, starch, sugar, glucose, methyl cellulose, magnesium stearate,mannitol, sorbitol, and the like. Exemplary liquid oral excipientsinclude ethanol, glycerol, water, and the like. Starch,polyvinyl-pyrrolidone (PVP), sodium starch glycolate, microcrystallinecellulose, and alginic acid are exemplary disintegrating agents. Bindingagents may include starch and gelatin. The lubricating agent, ifpresent, may be magnesium stearate, stearic acid or talc. If desired,the tablets may be coated with a material such as glyceryl monostearateor glyceryl distearate to delay absorption in the gastrointestinaltract, or may be coated with an enteric coating.

Capsules for oral administration include hard and soft gelatin capsules.To prepare hard gelatin capsules, active ingredient(s) may be mixed witha solid, semi-solid, or liquid diluent. Soft gelatin capsules may beprepared by mixing the active ingredient with water, an oil such aspeanut oil or olive oil, liquid paraffin, a mixture of mono anddi-glycerides of short chain fatty acids, polyethylene glycol 400, orpropylene glycol.

Liquids for oral administration may be in the form of suspensions,solutions, emulsions or syrups or may be lyophilized or presented as adry product for reconstitution with water or other suitable vehiclebefore use. Such liquid compositions may optionally contain:pharmaceutically-acceptable excipients such as suspending agents (forexample, sorbitol, methyl cellulose, sodium alginate, gelatin,hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel andthe like); non-aqueous vehicles, e.g., oil (for example, almond oil orfractionated coconut oil), propylene glycol, ethyl alcohol, or water;preservatives (for example, methyl or propyl p-hydroxybenzoate or sorbicacid); wetting agents such as lecithin; and, if desired, flavoring orcoloring agents.

The active agents of this invention may also be administered by non-oralroutes. For example, compositions may be formulated for rectaladministration as a suppository. For parenteral use, includingintravenous, intramuscular, intraperitoneal, or subcutaneous routes, theagents of the invention may be provided in sterile aqueous solutions orsuspensions, buffered to an appropriate pH and isotonicity or inparenterally acceptable oil. Suitable aqueous vehicles include Ringer'ssolution and isotonic sodium chloride. Such forms may be presented inunit-dose form such as ampules or disposable injection devices, inmulti-dose forms such as vials from which the appropriate dose may bewithdrawn, or in a solid form or pre-concentrate that can be used toprepare an injectable formulation. Illustrative infusion doses rangefrom about 1 to 1000 μg/kg/minute of agent admixed with a pharmaceuticalcarrier over a period ranging from several minutes to several days.

For topical administration, the agents may be mixed with apharmaceutical carrier at a concentration of about 0.1% to about 10% ofdrug to vehicle. Another mode of administering the agents of theinvention may utilize a patch formulation to affect transdermaldelivery.

Active agents may alternatively be administered in methods of thisinvention by inhalation, via the nasal or oral routes, e.g., in a sprayformulation also containing a suitable carrier.

Exemplary active agents useful in methods of the invention will now bedescribed by reference to illustrative synthetic schemes for theirgeneral preparation below and the specific examples that follow.

The compounds as described above may be made according to processeswithin the skill of the art and/or that are described in the schemes andexamples that follow. Certain reaction schemes may occur with or withoutprotection as appropriate. This may be achieved by means of conventionalprotecting groups, such as those described in “Protective Groups inOrganic Chemistry”, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W.Greene & P. G. M. Wuts, “Protective Groups in Organic Synthesis”, 3^(rd)ed., John Wiley & Sons, 1999. The protecting groups may be removed at aconvenient subsequent stage using methods known from the art.Alternatively, it may be necessary to employ, in the place of theultimately desired substituent, a suitable group that may be carriedthrough the reaction scheme and replaced as appropriate with the desiredsubstituent. Such compounds, precursors, or prodrugs are also within thescope of the invention.

In order to illustrate the invention, the following examples areincluded. These examples do not limit the invention. They are only meantto suggest a method of practicing the invention. Those skilled in theart may find other methods of practicing the invention, which areobvious to them. However, those methods are deemed to be within thescope of this invention.

The synthesis of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide will now be described by reference toillustrative synthetic schemes and a specific protocol for itspreparation. Artisans will recognize that, to obtain the variouscompounds herein, starting materials may be suitably selected so thatthe ultimately desired substituents will be carried through the reactionscheme with or without protection as appropriate to yield the desiredproduct. Alternatively, it may be necessary or desirable to employ, inthe place of the ultimately desired substituent, a suitable group thatmay be carried through the reaction scheme and replaced as appropriatewith the desired substituent.

Referring to Scheme A, Intermediate 1 was obtained by reacting2,2-difluoro-benzo[1,3]dioxole-5-carbaldehyde with piperazine underhydrogenation conditions. The reaction can be carried out using Pd(OH)₂,Pt, or Pd as a catalyst, in solvents such as MeOH, EtOH, or AcOH. Thereaction can be performed at temperatures between 20 to 80° C.Acceptable H₂ pressure may be between 1 to 60 bars. The amount of2,2-difluoro-benzo[1,3]dioxole-5-carbaldehyde to piperazine is typicallyone to six equivalents. The reaction can be performed on abatch-hydrogenation apparatus or a flow-hydrogenation apparatus.

Referring to Scheme B,4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide was prepared from3-amino-4-chloropyridine and Intermediate 1. 3-Amino-4-chloropyridinewas treated with phenyl chloroformate and pyridine in a solvent such astoluene to give the compound of Intermediate 2. Intermediate 2 wasdirectly reacted with Intermediate 1 to give4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide.

Chemistry:

In preparing4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide and the comparator example below, thefollowing general experimental and analytical methods were used.

Reaction mixtures were stirred under a nitrogen atmosphere unlessotherwise noted. Where solutions or mixtures are concentrated, they aretypically concentrated under reduced pressure using a rotary evaporator.

Mass spectra were obtained on an Agilent series 1100 MSD usingelectrospray ionization (ESI) in positive mode unless otherwiseindicated.

NMR spectra were obtained using either a Bruker model DPX400 (400 MHz),DPX500 (500 MHz), DRX600 (600 MHz) spectrometer. The format of the ¹HNMR data below is: chemical shift in ppm down field of thetetramethylsilane reference (multiplicity, coupling constant J in Hz,integration).

Chemical names were generated using ChemDraw Ultra 6.0.2 (CambridgeSoftCorp., Cambridge, Mass.) or ACD/Name Version 9 (Advanced ChemistryDevelopment, Toronto, Ontario, Canada).

Intermediate 1: 1-(2,2-Difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine

A 2 L Erlenmeyer flask was charged with piperazine (185.1 g, 2.15 mol),2,2-difluoro-benzo[1,3]dioxole-5-carbaldehyde (100.0 g, 0.537 mol), andmethanol (1.08 L). The solution was stirred at room temperature for 18 hthen passed twice through an H-Cube Midi™ (ThalesNano, Budapest,Hungary) with a new 20% Pd(OH)₂/C MidiCart cartridge, at the followingsettings: 70° C., 1 atm pressure, 6 mL/min flow rate, and 10% excess H₂production. The aldehyde starting material was >90% consumed after thefirst pass, and completely consumed after the second pass as indicatedby HPLC analysis. The methanol was evaporated, toluene (1.20 L) wasadded and the mixture was stirred at room temperature for 18 h. Theresulting white suspension was filtered and the solid was rinsed withtoluene (200 mL). The combined filtrate was washed with water (2×300mL), dried over Na₂SO₄, filtered, and concentrated to afford the productas a colorless oil. The oil was dissolved in heptane (100 mL) and theproduct was allowed to crystallize at room temperature. For subsequentsynthesis experiments, addition of a small amount of seed crystals wasused to quicken the crystallization process. The suspension was cooledto 0° C., filtered and the solid was dried in vacuum oven at 50° C. for24 h to give the title compound as a white solid (108.0 g, 78%). Theheptane filtrate was concentrated to approximately 20 mL followed byaddition of product seed crystals. The solution then was stirredovernight. The second batch of the title compound was obtained as awhite solid after filtration and drying (6.7 g, 5%). The combined yieldwas (115 g, 83%). MS (ESI⁺): calcd for C₁₂H₁₄F₂N₂O₂ m/z 256.1. found256.9 (M+H)⁺. ¹H NMR (400 MHz, CDCl₃) δ: 7.11 (d, J=0.9 Hz, 1H), 6.99(dd, J=9.0, 0.9 Hz, 1H), 6.95 (d, J=9.0 Hz, 1H), 3.45 (s, 2H), 2.92-2.83(m, 4H), 2.39 (s, 4H); ¹³C NMR (101 MHz, CDCl₃) δ: 143.89, 142.72,134.68, 131.65 (t, JC-F=255.3), 123.88, 110.13, 108.82, 63.10, 54.38,46.07.

EXAMPLE 14-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide

A 2 L, three-neck Morton flask equipped with a mechanical stirrer,thermocouple, and addition funnel under a nitrogen atmosphere wascharged with 3-amino-4-chloropyridine (35.0 g, 272 mmol) and toluene(740 mL). The brown solution was cooled to 2° C. Pyridine (25.3 mL, 310mmol) was added in one portion, followed by the dropwise addition ofphenyl chloroformate (32.6 mL, 259 mmol) over 30 min. The maximuminternal temperature was 5° C. After stirring at 2-5° C. for 7 h thereaction mixture became a thick yellow suspension. A cooled solution ofK₂CO₃ (53.6 g, 388 mmol) in water (216 mL) was added over 3 min, duringwhich the maximum internal temperature was 6° C.1-(2,2-Difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine (66.3 g, 259mmol) was then added as a solid over 1 min. The mixture was allowed towarm slowly to room temperature and stirred for 15 h. Water (200 mL) wasadded and the toluene layer was separated and extracted with aqueous HCl(1.8 M, 600 mL). The aqueous extract was washed with toluene (2×300 mL).MeOH (500 mL) was added to the aqueous layer and the solution was cooledto 5° C. The pH was adjusted to pH 8-9 with the addition of NaOHsolution (50 wt %, ca. 50 mL). The addition was at such a rate that theinternal temperature did not exceed 17° C. The resulting suspension wasstirred at 5° C. for 2 h. The product was collected by filtration andrinsed with MeOH/H₂O (1:1, 70 mL). The solid was dried in vacuum oven at50° C. for 24 h to afford the title compound as a yellow/green solid (73g, 69%).

A 1 L, three-neck Morton flask equipped with a stir bar, thermocouple,and reflux condenser was charged with crude4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide (98 g, 239 mmol) and isopropylacetate (318 mL). The suspension was heated to 65° C., treated withactivated charcoal (10.0 g) and stirred at 65° C. for 1 h. The mixturewas then heated to 80° C. and quickly filtered through a thin celitepad. The filtrate was slowly cooled to room temperature and then placedin an ice bath for 30 min. The solid was collected by filtration, rinsedwith cold iPrOAc (10 mL), and dried to give product4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide as a yellow solid (72 g, 73%).

A 2 L, three-neck Morton flask equipped with a mechanical stirrer,thermocouple, and reflux condenser was charged with crude product4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethylypiperazine-1-carboxylic acid(4-chloro-pyridin-3-yl)-amide (191 g, 465 mmol) and isopropyl acetate(705 mL). The suspension was heated to 65° C., treated with activatedcharcoal (11.2 g) and stirred at 65° C. for 1 h. The mixture was thenheated to 75° C. and quickly filtered. The filtrate was slowly cooled toroom temperature overnight and then placed in an ice bath for 30 min.The solid was collected by filtration, rinsed with cold iPrOAc (40 mL),and dried in vacuum oven at 50° C. for 72 h. The product4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide was obtained as a slightly yellowsolid (161 g, 84%). MS (ESI⁺): calcd for C₁₈H₁₇ClF₂N₄O₃ m/z 410.1, found411.1 (M+H)⁺. Anal. Calcd for C₁₈H₁₇ClF₂N₄O₃: C, 52.63; H, 4.17; N,13.64. Found: C, 52.73; H, 4.15; N, 13.62; ¹H NMR (600 MHz, CDCl₃) δ:9.36 (s, 1H), 8.19 (d, J=5.2 Hz, 1H), 7.29 (dd, J=5.3, 0.3 Hz, 1H), 7.13(d, J=0.9 Hz, 1H), 7.02-6.98 (m, 2H), 6.84 (s, 1H), 3.58-3.54 (m, 4H),3.53 (s, 2H), 2.54-2.48 (m, 4H); ¹³C NMR (151 MHz, CDCl₃) δ: 153.49,144.02, 143.88, 143.28, 142.98, 134.05, 133.09, 131.66 (t, JC-F=254.6Hz), 131.55, 123.89, 123.53, 110.03, 109.02, 62.28, 52.47, 44.23.

EXAMPLE 1A4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide, bis hydrochloride

A solution consisting of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide (5.0 g, 12 mmol) and ethanol (200 mL)was treated with saturated aqueous HCl (3.0 mL, 3 equiv). The solventwas removed in vacuo, ethanol (100 mL) was added and the suspension wascooled to 0° C. and filtered. The resulting white solid was rinsed withcold ethanol (25 mL) and dried under vacuum to give4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide bis-hydrochloride (4.25 g, 72%). ¹HNMR (400 MHz, DMSO) δ 11.61 (br s, 1H), 8.98 (s, 1H), 8.65 (s, 1H), 8.37(d, J=5.4 Hz, 1H), 7.78 (d, J=1.4 Hz, 1H), 7.69 (d, J=5.4 Hz, 1H), 7.52(d, J=8.3 Hz, 1H), 7.46 (dd, J=8.3, 1.5 Hz, 1H), 4.39 (s, 2H), 4.28-4.12(m, 2H), 3.49-3.26 (m, 4H), 3.03 (s, 2H).

Comparator Compound:4-(2,2-Difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid pyridin-3-ylamide

Pyridin-3-yl-carbamic acid phenyl ester: To a solution consisting ofpyridin-3-yl amine (9.49 g, 101 mmol) and pyridine (8.77 g, 111 mmol) inCH₃CN (80 mL) at 0° C. was added phenyl chloroformate (15.8 g, 101 mmol)dropwise. The reaction mixture was allowed to warm to room temperatureand stirred for 2 h. The reaction was quenched with H₂O (200 mL) and theresulting precipitate was filtered and dried under vacuum to provide thetitle compound as a tan solid (17.34 g, 80%). MS (ESI⁺): calcd forC₁₂H₁₀N₂O₂ m/z 214.07, found 215.3 (M+H)⁺. ¹H NMR (500 MHz, d⁶-DMSO):10.46 (s, 1H), 8.69 (d, J=2.4 Hz, 1H), 8.27 (dd, J=4.7, 1.4 Hz, 1H),7.93 (d, J=8.4 Hz, 1H), 7.47-7.41 (m, 2H), 7.37 (dd, J=8.4, 4.7 Hz, 1H),7.31-7.22 (m, 3H).

4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid pyridin-3-ylamide: To a solution of pyridine-3-yl-carbamic acidphenyl ester (9.08 g, 42.4 mmol) in DMSO (84 mL) was added1-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine (11.4 g, 44.5mmol). The reaction mixture was stirred at room temperature for 16 h,then treated with water (130 mL). The resulting solid was isolated byfiltration, rinsed with water (4×50 mL) and dried under vacuum. Thesolid was recrystallized (EtOH—H₂O) to give4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid pyridin-3-ylamide (13.4 g, 84%). MS (ESI⁺): calcd for C₁₈H₁₈F₂N₄O₃m/z 376.13. found 377.1 (M+H)⁺. ¹H NMR (CDCl₃): 8.46 (d, J=2.5 Hz, 1H),8.23-8.21 (m, 1H), 7.99-7.96 (m, 1H), 7.22-7.19 (m, 2H), 7.11 (s, 1H),6.99-6.98 (m, 2H), 3.54-3.52 (m, 4H), 3.49 (s, 2H, 2.46-2.44 (m, 4H).

Biological Testing:

-   -   Assay Method 1

A. Transfection of Cells with Human FAAH

A 10-cm tissue culture dish with a confluent monolayer of SK-N-MC cellswas split 2 days (d) prior to transfection. Using sterile technique, themedia was removed and the cells were detached from the dish by theaddition of trypsin. One fifth of the cells were then placed onto a new10-cm dish. Cells were grown in a 37° C. incubator with 5% CO₂ inMinimal Essential Media Eagle with 10% Fetal Bovine Serum. After 2 d,cells were approximately 80% confluent. These cells were removed fromthe dish with trypsin and pelleted in a clinical centrifuge. The pelletwas re-suspended in 400 μL complete media and transferred to anelectroporation cuvette with a 0.4 cm gap between the electrodes.Supercoiled human FAAH cDNA (1 μg) was added to the cells and mixed. Thevoltage for the electroporation was set at 0.25 kV, and the capacitancewas set at 960 μF. After electroporation, the cells were diluted intocomplete media (10 mL) and plated onto four 10-cm dishes. Because of thevariability in the efficiency of electroporation, four differentconcentrations of cells were plated. The ratios used were 1:20, 1:10,and 1:5, with the remainder of the cells being added to the fourth dish.The cells were allowed to recover for 24 h before adding the selectionmedia (complete media with 600 μg/mL G418). After 10 d, dishes wereanalyzed for surviving colonies of cells. Dishes with well-isolatedcolonies were used. Cells from individual colonies were isolated andtested. The clones that showed the most FAAH activity, as measured byanandamide hydrolysis, were used for further study.

B. FAAH Assay

T84 frozen cell pellets or transfected SK-N-MC cells (contents of 1×15cm culture dishes) were homogenized in 50 mL of FAAH assay buffer (125mM Tris, 1 mM EDTA, 0.2% Glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH9). The assay mixture consisted of 50 μL of the cell homogenate, 10 μLof the test compound, and 40 μL of anandamide [1-³H-ethanolamine](³H-AEA, Perkin-Elmer, 10.3 C_(i)/mmol), which was added last, for afinal tracer concentration of 80 nM. The reaction mixture was incubatedat rt for 1 h. During the incubation, 96-well Multiscreen filter plates(catalog number MAFCNOB50; Millipore, Bedford, Mass., USA) were loadedwith 25 μL of activated charcoal (Multiscreen column loader, catalognumber MACL09625, Millipore) and washed once with 100 μL of MeOH. Alsoduring the incubation, 96-well DYNEX MicroLite plates (catalog numberNL510410) were loaded with 100 μL of MicroScint40 (catalog number6013641, Packard Bioscience, Meriden, Conn., USA). After the 1 hincubation, 60 μL of the reaction mixture were transferred to thecharcoal plates, which were then assembled on top of the DYNEX platesusing Centrifuge Alignment Frames (catalog number MACF09604, Millipore).The unbound labeled ethanolamine was centrifuged through to the bottomplate (5 min at 2000 rpm), which was preloaded with the scintillant, asdescribed above. The plates were sealed and left at rt for 1 h beforecounting on a Hewlett Packard TopCount.

-   -   Assay Method 2

A. Transfection of Cells with Rat FAAH

A 10-cm tissue culture dish with a confluent monolayer of SK-N-MC cellswas split 2 days (d) prior to transfection. Using sterile technique, themedia was removed and the cells were detached from the dish by theaddition of trypsin. One fifth of the cells were then placed onto a new10-cm dish. Cells were grown in a 37° C. incubator with 5% CO₂ inMinimal Essential Media Eagle with 10% Fetal Bovine Serum. After 2 d,cells were approximately 80% confluent. These cells were removed fromthe dish with trypsin and pelleted in a clinical centrifuge. The pelletwas re-suspended in 400 μL complete media and transferred to anelectroporation cuvette with a 0.4 cm gap between the electrodes.Supercoiled rat FAAH cDNA (1 μg) was added to the cells and mixed. Thevoltage for the electroporation was set at 0.25 kV, and the capacitancewas set at 960 μF. After electroporation, the cells were diluted intocomplete media (10 mL) and plated onto four 10-cm dishes. Because of thevariability in the efficiency of electroporation, four differentconcentrations of cells were plated. The ratios used were 1:20, 1:10,and 1:5, with the remainder of the cells being added to the fourth dish.The cells were allowed to recover for 24 h before adding the selectionmedia (complete media with 600 μg/mL G418). After 10 d, dishes wereanalyzed for surviving colonies of cells. Dishes with well-isolatedcolonies were used. Cells from individual colonies were isolated andtested. The clones that showed the most FAAH activity, as measured byanandamide hydrolysis, were used for further study.

B. FAAH Assay

Transfected SK-N-MC cells (contents of 1×15 cm culture dishes) werehomogenized in 50 mL of FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2%Glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9). The assay mixtureconsisted of 50 μL of the cell homogenate, 10 μL of the test compound,and 40 μL of anandamide [1-³H-ethanolamine] (³H-AEA, Perkin-Elmer, 10.3C_(i)/mmol), which was added last, for a final tracer concentration of80 nM. The reaction mixture was incubated at rt for 1 h. During theincubation, 96-well Multiscreen filter plates (catalog number MAFCNOB50;Millipore, Bedford, Mass., USA) were loaded with 25 μL of activatedcharcoal (Multiscreen column loader, catalog number MACL09625,Millipore) and washed once with 100 μL of MeOH. Also during theincubation, 96-well DYNEX MicroLite plates (catalog number NL510410)were loaded with 100 μL of MicroScint40 (catalog number 6013641, PackardBioscience, Meriden, Conn., USA). After the 1 h incubation, 60 μL of thereaction mixture were transferred to the charcoal plates, which werethen assembled on top of the DYNEX plates using Centrifuge AlignmentFrames (catalog number MACF09604, Millipore). The unbound labeledethanolamine was centrifuged through to the bottom plate (5 min at 2000rpm), which was preloaded with the scintillant, as described above. Theplates were sealed and left at rt for 1 h before counting on a HewlettPackard TopCount.

Results for compounds tested in these assays are summarized in Table 1,as an average of results obtained. Compounds were tested in either theirfree base or hydrochloride salt form. The comparator compound,4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid pyridin-3-ylamide, was synthesized as described in PCT. Pub. Appl.No. WO 2006/074025, example 150.

TABLE 1 Assay 1 Assay 2 Compound IC₅₀ (nM) IC₅₀ (nM) Example 1  75 320Comparator Compound 340 450Drug-Drug Interaction (DDI) Assay

The potential for4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide and Comparator Compound,4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid pyridin-3-ylamide, to inhibit human cytochrome P-450 isoenzymes(CYPs) was investigated by incubating the compound at variousconcentrations with human liver microsomes and specific CYP probesubstrates (Table 2). CYP inhibition can impact the safety profile of adrug substance by interfering with the metabolism of other drugmolecules.

The assay was set up and executed using a Biomek FXp robotic liquidhandling workstation (Beckman Coulter Corp., Fullerton, Calif.),integrated with a Cytomat shaking incubator set at 37° C. (ThermoElectron Corp., Bellefonte, Pa.). A batch of human liver microsomes from50 donors, pooled and characterized by BD Gentest, (Cat #457111, lot01220, 20 mg/mL in 250 mM sucrose) was used. Each substrate wasincubated at a protein concentration of 0.1, 0.15 or 0.2 mg/mL in atotal incubation volume of 0.16 mL. The incubates were prepared in 100mM potassium phosphate buffer (pH 7.4) supplemented with 5 mM magnesiumchloride and 1 mM EDTA. Quinidine was used as a positive controlinhibitor for CYP2D6. Quinidine was prepared as a working solution inorganic solvent (primarily methanol, with DMSO and acetonitrile assecondary solvents) and was spiked into the microsomal suspension toyield the desired concentration level. The solution was then seriallydiluted with additional microsomal suspension to yield eightconcentration levels. Final organic content was less than 0.07%. A stocksolution of the test compound was prepared at a concentration of 50 mMor higher, if possible, in an adequate organic solvent (DMSO, methanolor acetonitrile), depending on solubility limitations. The stocksolution was serially diluted with methanol and subsequently spiked intothe microsomal suspension to yield final incubation concentrations of 0,0.1, 0.3, 1, 3, 10, 30 and 100 μM for Comparator Compound and 0, 0.06,0.18, 0.6, 1.8, 6, 18 and 60 μM for4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide. The final organic content was 0.2%.Incubations were performed in triplicate for each probe substrate.

The control inhibitor (quinidine) and marker substrate (dextromethorphanor bufuralol) were transferred to the incubation vessels (60 μL aliquotseach). After a pre-incubation period at 37° C., the reactions wereinitiated by the addition of a 40 μL aliquot of NADPH regeneratingsystem (BD Gentest). A 40 μL aliquot (diluted 6:19 with incubationbuffer) provided final concentrations of 1.3 mM NADP+, 3.3 mMglucose-6-phosphate and 0.4 U/mL glucose-6-phosphate dehydrogenase.Incubation times were 12 minutes for both dextromethorphan andbufuralol. Reactions were terminated by the direct addition ofacetonitrile (160 μL) to the incubation mix followed by transfer to apooling plate containing additional acetonitrile (400 μL).

The incubation reactions were pooled by equal test compound or controlinhibitor concentration, transferred to a Phenomenex Strata™ Impactprotein precipitation filter plate containing acetonitrile and internalstandards (100 μL of a mixture of the following deuterated compoundsranging in concentration from 0.5 to 2.8 μM: hydroxybufuralol-d₉,dextrorphan-d₃). The resulting filtrate was evaporated to dryness undera nitrogen flow, then reconstituted in 250 μL mobile phase (1:1methanol:water, containing 0.1% acetic acid). Samples and standards wereanalyzed on a Sciex API4000 triple quadrupole mass spectrometer. Thedata were acquired in Analyst 1.4.1 (Applied Biosystems/MDS Sciex). ForIC₅₀ assays, the area ratios of the metabolite and internal standardchromatographic peaks were transferred to SigmaPlot (version 8.0) andplotted on a semi-log scale (percent residual activity vs. inhibitorconcentration) to determine the IC₅₀ value.

4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide inhibited CYP2D6 with an IC₅₀ valueof 24 μM using Bufuralol as the test substrate, and 11 μM usingDextromorphan as the test substrate.4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide compared favorably to ComparatorCompound, which inhibited CYP2D6 with an IC₅₀ value of 3.2 μM usingBufuralol, and 2.0 μM using Dextromorphan as the test substrates. Theseresults demonstrate an potential reduced risk of drug-drug interactionsfor 4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide relative to Comparator Compound.

TABLE 2 2D6 2D6 Compound Bufuralol (μM) Dextromethorphan (μM) Example 124 11 Comparator Compound 3.2 2.0Primary Observation (Irwin) Test in the Rat

4-(2,2-Difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide hydrochloride and the hydrochloridesalt of Comparator Compound were investigated with oral administrationin the Primary Observation (Irwin) Test in the rat to evaluate theirgeneral effects on behavior and physiological functions. The method,which detects the first toxic dose, the active dose-range and the maineffects of a test substance on behavior and physiological function,follows that described by Irwin et al., Psychopharmacologia, 1968, 13,222-257. The rats were administered the test substance and were observedin simultaneous comparison with a control group given vehicle (non-blindconditions). All animals within a treatment group were observedsimultaneously. Behavioral modifications, physiological andneurotoxicity symptoms, rectal temperature and pupil diameter wererecorded according to a standardized observation grid derived from thatof Irwin. The grid contains the following items: death, convulsions,tremor, Straub tail, altered activity, jumping, abnormal gait (rolling,tip-toe), motor uncoordination, altered abdominal muscle tone, loss ofgrasping, akinesia, catalepsy, loss of traction, loss of balance,fore-paw treading, writhes, piloerection, stereotypies (sniffing,chewing, head movements), head twitches, scratching, alteredrespiration, aggression, altered fear/startle, altered reactivity totouch, ptosis, exophthalmia, loss of righting reflex, loss of cornealreflex, analgesia, defecation/diarrhea, salivation, lacrimation, rectaltemperature (hypothermia/hyperthermia) and pupil diameter(myosis/mydriasis). The test substances were evaluated at 2 doses (10and 60 mg/kg), administered p.o. immediately before the test, andcompared with a vehicle control group. Observations were performed 15,30, 60, 120 and 180 minutes after administration of the testsubstances/vehicles and also 24 hours later.

TABLE 3 Comparator Compound HCl Example 1 (mg/kg p.o.) (mg/kg p.o.) 1060 10 60 Increased Sedation: Increased Sedation: reactivity (0/4)reactivity (3/3) 15 to 120 min to touch: Abnormal gait: to touchAbnormal gait (1/4) at (0/4) (3/3) at 15 (rolling): 180 min Hypothermia:min (3/3) at 15 min (0/4) Hypothermia: Increased slight induction at 15abdominal muscle to 60 min and at 180 tone: min (1/4) at 60 minDecreased muscle Increased reactivity tone: to touch: (1/3) at 30 min(1/4) at 60 min (3/3) at 60 to 120 (1/4) at 180 min min Increasedreactivity to touch: (0/3) at 60 min and 180 min

4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide hydrochloride caused weak andtransient arousing effects in only one rat over the dose-range 10-60mg/kg in the Irwin Test (Table 3). At 10 mg/kg,4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide hydrochloride increased reactivity totouch in 1 out of 4 rats at 180 minutes. At 60 mg/kg,4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide hydrochloride increased abdominalmuscle tone in 1 out of 4 rats at 60 minutes and increased reactivity totouch in 1 out of the 4 rats at 60 and 180 minutes. Apart from transientand occasional increase in abdominal muscle tone at 60 mg/kg, no othersigns were observed up to 24 hours after administration.

4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide hydrochloride showed favorabledifferentiation from Comparator Compound HCl in the Irwin Test at bothdoses. Whereas4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide hydrochloride caused increasedreactivity to touch in only 1 in 4 rats at 10 mg/kg, Comparator CompoundHCl increased reactivity to touch in all 3 rats. At 60 mg/kg, ComparatorCompound HCl induced sedation from 15 to 120 minutes and abnormal gait(rolling) at 15 minutes in all 3 rats. It decreased muscle tone in 1 ratat 30 minutes and from 60 to 120 minutes in all 3 rats. It also inducedhypothermia from 15 to 60 minutes and at 180 minutes. These resultssuggest the presence of sedative/depressant effects (sedation, motorsigns and hypothermia) for Comparator Compound HCl at 60 mg/kg p.o. thatwere not observed with4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide hydrochloride.

What is claimed is:
 1. A compound that is4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide or a pharmaceutically acceptable saltthereof.
 2. The pharmaceutically acceptable salt of claim 1, whereinsaid salt is a hydrochloride salt of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)amide.
 3. The pharmaceutically acceptablesalt of claim 2, wherein said hydrochloride salt is bis-hydrochloride.4. A pharmaceutical composition comprising: (a) a therapeuticallyeffective amount of4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide or a pharmaceutically acceptable saltthereof; and (b) a pharmaceutically acceptable excipient.
 5. A method oftreating pain, comprising administering to a subject in need of suchtreatment a therapeutically effective amount of a compound as defined inclaim
 1. 6. A method of synthesizing4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylicacid (4-chloro-pyridin-3-yl)-amide using2,2-difluoro-benzo[1,3]dioxole-5-carbaldehyde and piperazine in a singlestep hydrogenation reaction.